Manufacture and purification of cyclosporin A

ABSTRACT

The present invention provides an improved method for the manufacture of Cyclosporin A is disclosed. Cyclosporin A is produced by solid state fermentation of  Fusarium solani  in a bioreactor under optimal fermentation parameters. The product is extracted and further purified by treating with alum and subsequent chromatographic procedures to get pharmaceutical acceptable purity.

FIELD OF THE INVENTION

The present invention relates to an improved method for the manufactureand purification of Cyclosporin A (CsA).

BACKGROUND

CsA is a member of a group of cyclic undecapeptides withanti-inflammatory, immunosuppressive, antifungal and antiparasiticproperties. The immunomodulatory properties are of particular interestin medicine, since they are important in prevention of organ rejectionin transplantation surgery and for autoimmune diseases.

Cyclosporins are a class of compounds which were first described inSwiss patents 589716 and 603790. They have been reviewed in themonograph by Borel [Cyclosporin, Progress in Allergy (1986) 38, KargerPress] discussing the chemical structure and thebiological-pharmacological properties.

Over 20 different cyclosporins are produced by fungi all having varyingamino acid composition. CsA is the major component and currently enjoysthe most widespread clinical use.

In extensive screening experiments, numerous fungal species were foundto have CsA forming properties. Dreyfuss [Sydowia (1986) 39, 22–36]described an exclusively Cyclosporin forming ability for Cylindrocarponand Fusarium. Cyclosporin formation is also reported fromfollowing—Tolypocladium geodes, Trichoderma virile, Neocosmosporavasunfecta, Isaria spp., Verticillium spp., Acremonium spp. andBeauveria nivea.

Sesquicilliopsis rosariensis F 605 with 3150 mg/L after 14 days ofsubmerged fermentation and Tolypocladium inflatum Wb 6-5 with 1100 mg/Lafter 11 days of fermentation are known to be high producers (U.S. Pat.No. 5,256,547, 1993). Tolypocladium inflatum KD 461, a mutant whensupplemented with L-leucine and L-valine produced 8920 mg/L after 13days (WO Patent No. 96/12032, 1996).

Tolypocladium inflatum strains have been used in solid statefermentation for Cyclosporin fermentations. Strains producing 1.95 g/kgto 4.84 g/kg of wheat bran during 5–10 days of fermentation have beenreported [Balaraman and Mathew EP No. 0 725 076 (1996); Sekar andBalaraman, Ind. J. Microbiol. (1996) 36, 231–234; Sekar et al,Bioprocess Engg. (1997) 18, 257–259; Murthy et al, Process Biochem.(1999) 34, 269–280].

The economics of using a producing strain, the describedvolume-time-yields in the above process are not satisfactory. Further,there are difficulties of maintaining a low temperature of 25° C. Thereare also disadvantages in isolation and purification of differentcyclosporins in this process.

The patent specification EP 0 725 076, also discloses a process for themanufacture of CsA by solid state fermentation—but uses a Tolypocadiumsp. and an uncontained system. Since the fermentation process disclosedin EP 0725 076 is not contained it doesn't assure full safety for thecytotoxic fermentation products like CsA—making the process industriallyunattractive.

The purification processes to isolate pharmacopoeial grade CsA usedconventionally are extraction of fermented biomass using an organicsolvent, evaporation of solvent, reextraction of residue, concentrationand then subjecting the residue to various chromatographic processes toseparate CsA from other cyclosporins and impurities viz., gel filtrationtypically LH-20 (U.S. Pat. Nos. 4,117,118 and 5,256,547) and/or silicagel or alumina columns (U.S. Pat. No. 4,117,118) or adsorption resincolumns (patent No. WO 97/46575). Final yields obtained from theconventional processes are typically in the range of 40% to 60%. Removalof the lipids from the fermented media is done by using pet ether,hexane, acetone, methanol or a mixture of these (British patent No.GB2,227,489). CsA being mixable in these lipids as well as in the mixtureof acetone and petroleum ether, a significant amount of CsA is lostduring this step.

Super critical fluid extraction has also been reported to have been usedfor separation of cyclosporin using super critical carbon dioxide(Canadian patent No. CA 2,108,655). This process has economiclimitations for its commercial application.

DESCRIPTION OF THE INVENTION

The objects of this invention therefore are:

-   -   to provide a novel bioreactor for a contained solid state        fermentation such that the fermenting micro-organism and the        fermentation products it produces are kept isolated from the        outside environment during the course of fermentation    -   to reduce solvent consumption required in large quantities for        extracting CsA from fermentation broth or the aqueous residue    -   to produce higher yield and purity of CsA    -   to assure full safety for the cytotoxic fermentation products        like CsA.    -   to reduce the period for fermentation and to control the        temperature easily.    -   To achieve the said objectives this invention provides an        improved method for the manufacture and purification of        cyclosporin A comprising:    -   loading a contained bioreactor with solid state nutritive matrix        and sterilizing it,    -   mixing the said sterilized solid state nutritive matrix with        Fusarium sp.,    -   incubating the said inoculated solid state nutritive matrix for        4–6 days at 25–30° C.,    -   extracting the fermented matrix with an organic solvent,    -   concentrating the organic solvent extract and treating with 0.1        to 5.0% alum for 2 to 6 hours to obtain CsA precipitate,        followed by filtration using a filter aid,    -   dissolving the said filter aid cake in a hydrocarbon organic        solvent and concentrating to get a residue,    -   subjecting the said residue dissolved in an alcohol to gel        filtration,    -   pooling the CsA containing fractions and subjecting to silica        gel chromatography, and removing the organic solvent by        distillation,    -   dissolving the solid obtained in 2 to 6 times the volume of an        organic solvent and allowing it to chill at −10 to −45° C. for 2        to 8 hours.    -   filtering the organic solvent to get pure cyclosporin.

The said Fusarium sp. is Fusarium solani, which is in the form of amacroconidial suspension.

The said contained bioreactor allows solid state fermentation to becarried out in a manner such that the fermentation micro-organisms andthe fermentation products produced are kept isolated from the outsideenvironment during the course of fermentation. The said containedbioreactor is “PLAFRACTOR”.

The solid state nutritive matrix is selected from wheat bran, rice bran,soya grits or rice grits.

The organic solvent used for extraction is selected from acetone,methanol, toluene, benzene or ethyl acetate.

The alum used for treating the aqueous concentrate is either potash alumor ammonium alum.

The filter aid is selected from celite, perlite or alumina

The solvent used to dissolve the residue is selected from cyclohexane,toluene, benzene.

The gel filtration matrix is Sephadex LH-20.

The alcohol used as an eluant for gel filtration is selected frommethanol, ethanol or iso-propanol.

The mobile phase used is acetone, hexane or a mixture of these.

The residue obtained is dissolved in 2 times the volume of acetone andchilling at −20° C.

The novel contained bioreactor is ‘PLAFRACTOR’ and is capable ofsterilizing the solid state fermentation media, cooling it to therequired temperature, fermenting at the desired set conditions, in situextraction of the is end product, recovery of the solvents and postharvest sterilization (International publication no. WO 00/29544). The‘PLAFRACTOR’, a novel bioreactor for solid state fermentation is aunique invention of Biocon India Limited The modular construction ofthis bioreactor provides multiple modules stacked on top of one another,each with a base connected to frame for holding the solid medium inisolation from the exterior environment. The construction of thebioreactor allows solid state fermentation to be carried out in a mannersuch that the fermenting microorganisms and the fermentation products itproduces are kept isolated from the outside environment during thecourse of the fermentation. This containment of the fermentation processis of significant importance when working with microbial metabolites,which are cytotoxic in nature e.g. Cyclosporin. An important aspect ofthe bioreactor is a mechanism of heat removal resulting in stringenttemperature control of the fermentation process. In comparison,maintaining a constant temperature of growth in solid state fermentationusing tray cultures is not efficient. The base plate of the bioreactorhas multiple channels called noncommunicating channels that carryheating and cooling fluids sandwiched between two sheets. Heat istransferred to and from the modules by conduction. In this way thetemperature of the module is precisely maintained to meet the specificrequirement of different microorganisms.

The base of the module contains a second set of channels, thecommunicating channels to deliver sterile air as supply of oxygen intothe solid substrate bed for optimum growth of organism. Moisture lossbecause of passage of sterile air is significantly reduced by regularlyreversing the direction of airflow every few hours. Using this,homogeneity in moisture content is maintained throughout the bioreactor.These aspects provide ample convenience over previous SSF methodologiesthat require multiple manipulations at each step of the fermentationprocess.

The invention will now be described with reference to the followingexamples:

EXAMPLE 1

Heavily sporulated slants of Fusarium solani was taken and 5 mL ofsterile distilled water was added to it. It was shaken thoroughly and500 L of the spore suspension was spread on MEA (Malt Extract Agar)plates and incubated at 30° C. for 7 days. After 7 days the macroconidalodged as a slime is scraped by a sterile loop in sterile distilledwater. This macro conidial suspension, devoid of mycelial bits is usedas the inoculum. 15 Kg of wheat bran was loaded on the containedbioreactor of approximately 22600 cm² of plate area. The bioreactor wassterilized by sending steam simultaneously into the communicating andthe noncommunicating channels to heat the bioreactor and its contents toa temperature of 121° C. for 1–2 hours. The steam pressure was releasedand simultaneously sterile air was sent into the communicating channelswhile cooling water at approximately 30° C. was sent into thenoncommunicating channels.

The master seed for inoculation of culture was a 104 macroconidia/mlsuspension of Fusarium solani in 14 L of sterilized dist. water. Thiswas used to inoculate the sterilized wheat bran so that the finalmoisture after inoculation was 65%. The inoculum was mixed thoroughlywith the sterilized bran. Sterile airflow at a rate of 20 Lpm on thefirst day, 40 Lpm on second and third day and 20 Lpm on fourth and fifthday was sent into the bioreactor continuously. The temperature wascontrolled at 30° C. for all 5 days by conductive heating and cooling.The CsA production titres was assayed following extraction using theHPLC.

EXAMPLE 2

The contained bioreactor was sterilized and inoculated as in Example 1.In this experiment, the temperature was maintained at 25° C. for all 5days. The CsA production titres was assayed following extraction usingthe HPLC.

EXAMPLE 3

The contained bioreactor was sterilized and inoculated as in Example 1.In this experiment, rice bran was used instead of wheat bran and thetemperature was maintained at 30° C. for all 5 days. The CsA productiontitres was assayed following extraction using the HPLC.

EXAMPLE 4

5 Kg. fermented wheat bran obtained from Example 2 was then extracted byusing methanol. 10 L of methanolic extract was collected, analyzed andtaken for further processing. The extraction efficiency of methanol wasfound to be 98%, as quantitated by HPLC.

EXAMPLE 5

The extract obtained from Example 4 was concentrated by azeotropicdistillation to remove methanol, leaving behind 1.5 Its of aqueousresidue. To this aqueous residue, 2.5% (w/v) potassium alum was addedand allowed to stand for 3 hrs. After 3 hrs. precipitate of cyclosporinwas found floating at the surface of the liquid. This precipitate wasseparated by filtration through a CELITE (diatomaceous earth) bed.Recovery of cyclosporin by this precipitation was found to be 100%.Precipitate trapped on the CELITE (diatomaceous earth) bed wasredissolved completely in 400 ml of cyclohexane. Cyclohexane was removedby distillation to leave behind 17.55 gm of solid residue containing10.5 gm of CsA. This residue dissolved in methanol was subjected tofirst gel filtration using Sephadex LH-20 with methanol as an eluent.The fractions containing CsA were pooled and further subjected to silicagel (230–400 mesh) column chromatography, with acetone and hexanemixture (3:7) as the mobile phase. Fractions containing only CsA werepooled together and solid CsA was obtained by removing the solventscompletely by vacuum distillation. Desired quality of CsA crystals wereobtained by dissolving the solid CsA powder obtained from chromatographyin 3 times the volume of acetone and chilling at −20° C. Crystalsobtained after filtration were found to be of acceptable pharmaceuticalgrade purity as assayed by HPLC.

EXAMPLE 6

The extract obtained was processed as in Example 4. In this experimentBioreactor was sterilized and inoculated as in Example 2. In thisexperiment, 1.0% (w/v) of ammonium alum was used instead of potash alum.Crystals obtained after acetone crystallization were found to be ofacceptable pharmaceutical grade purity as assayed by HPLC.

The examples given above are not exhaustive.

The present invention has the following advantages over the otherreported methods

-   -   (i) Fermentation in a bioreactor, which is fully contained as a        result assuring full safety for the cytotoxic fermentation        products like CsA.    -   (ii) Less fermentation time and easier control of temperature        making the process economically attractive.    -   (iii) Fewer steps for the isolation and purification to get the        pure product, thus saving processing time and additional        expenses.    -   (iv) High efficiency of the alum treatment step during the        purification process results in higher yields.

1. A method for the manufacture and purification of cyclosporin A (CsA)comprising: loading a contained bioreactor with solid state nutritivematrix and sterilizing it, wherein the bioreactor comprises multiplestacked modules, each with a base connected to a frame, and wherein eachbase contains a plurality of communicating and noncommunicatingchannels, mixing the said sterilized solid state nutritive matrix withFusarium sp. used as inoculum, incubating the said inoculated solidstate nutritive matrix for 4–6 days at 25–30° C., extracting thefermented matrix with an organic solvent, concentrating the organicsolvent extract and treating with 0.1 to 5.0% alum for 2 to 6 hours toobtain a CsA precipitate, followed by filtration using a filter aid,dissolving the CsA precipitate in a hydrocarbon organic solvent andconcentrating to get a residue, treating the residue with alcohol andsubjecting the residue to gel filtration to produce CsA containingfractions, pooling the CsA containing fractions and subjecting to silicagel chromatography, elution and removing the organic solvent bydistillation to produce a concentrate, dissolving the concentrateobtained in 2 to 6 times the volume of an organic solvent and allowingto chill at −10 to −45° C. to 2 to 8 hours, and filtering the organicsolvent to get pure cyclosporin A.
 2. The method as claimed in claim 1wherein the Fusarium sp. used as inoculum is Fusarium solani.
 3. Themethod as claimed in claim 2 wherein the Fusarium solani used asinoculum is in the form of a macroconidial suspension.
 4. The method asclaimed in claim 1 wherein the said contained bioreactor allows solidstate fermentation to be carried out in a manner such that thefermentation microorganisms and the fermentation products produced arekept isolated from the outside environment during the course offermentation.
 5. The method as claimed in claim 1 wherein the solidstate nutritive matrix is wheat bran, rice bran, soya grits or ricegrits.
 6. The method as claimed in claim 1 wherein the organic solventused for extraction is acetone, methanol, toluene, benzene or ethylacetate.
 7. The method as claimed in claim 1 wherein the alum used fortreating the aqueous concentrate is either potash alum or ammonium alum.8. The method as claimed in claim 1 wherein the filter aid is(diatomaceous earth), perlite or alumina.
 9. The method as claimed inclaim 1 wherein the solvent used to dissolve the concentrate iscyclohexane, toluene or benzene.
 10. The method as claimed in claim 1wherein the alcohol used as an eluant for gel filtration is methanol,ethanol or iso-propanol.
 11. The method as claimed in claim 1 wherein aneluent is used for the silica gel chromatography is acetone, hexane or amixture thereof.
 12. The method as claimed in claim 1 wherein theconcentrate is dissolved in 2 times the volume of acetone and chilled at−20° C.